Uric acid detection



United States Patent 3,536,448 URIC ACID DETECTION ChittaranjanPurushottamdas Patel, Elkhart, Ind., as-

signor to Miles Laboratories, Inc., Elkhart, Ind., a corporation ofIndiana No Drawing. Filed July 28, 1967, Ser. No. 656,652 Int. Cl. Gllln21/20, 21/22, 33/16 US. Cl. 23-230 11 Claims ABSTRACT OF THE DISCLOSUREAn alkaline phosphotungstate process and a composition for detectinguric acid in which a composition comprising a color enhancer and anamino carboxylic sequestering agent is utilized in combination with aphosphotungstate reagent.

Background of the invention This invention relates to the detection ofuric acid and more particularly to a new process and novel compositionfor the detection of uric acid in fluids.

Uric acid is normally present in some quantities in the fluids ofmammals, particularly humans, as. a result of normal body functions.Concentrations of uric acid, especially in the blood and urine, provideeasily obtainable and correlatable indications of body conditions whichmay be adverse to general good health.

For example, ingestion of foods, such as purine-containing foods,normally has no effect on the uric acid blood level. An unexplainableevaluation of such concentration may, however, be indicative of renalinsufficiency. In some pathological conditions not related to dietaryingestion, such as uremia and gout, an abnormal increase occurs in theconcentration of uric acid in the blood. Unusual levels of uric acid inthe blood are also known to be associated with conditions that causeexcessive destruction of the nuclei of white blood corpuscles, forexample, lukemia and pneumonia.

Medical science has long recognized the usefulness of a test for uricacid as an aid in diagnosing the foregoing conditions, and in someinstances, distinguishing between closely related abnormal conditions,for example, gout and arthritis. Gout is characterized by an abnormalincrease in uric acid in the blood whereas arthritis does not exhibitsuch increase. For such reasons it has been considered desirable to havea test which is simple and inexpensive and yet provides a precisedetermination of uric acid concentration.

Uric acid is generally found in the blood in quantities varying betweenabout 0.7 and 7.0 milligrams per 100 milliliters, reported as mg.percent. This percent varies over a wide range as a result of exercise,diet and similar factors. With abnormal conditions, values of blood uricacid concentrations of the blood in excess of two and three times theirusual value often occur.

Two major processes have been developed for detecting uric acid and arebroadly described as the uricase method and the alkalinephosphotungstate method.

The uricase method is based on the principle that uric acid has anultraviolet absorption peak in the range of 290 to 293 m whereas the endproducts of the reaction of uricase and uric acid have no absorption atthis Wavelength. To determine uric acid, serum is reacted with a uricaseenzyme and the decrease in absorbance is measured. This method, whileproviding accurate determinations, requires highly skilled laboratorypersonnel, expensive equipment and a great deal of time to perform.

Alkaline phosphotungstate determination of uric acid was developed about1894 and has been subjected to ice many alterations since that time. Fordetection of uric acid in b ood with this procedure, phosphotungsticacid is added to a protein free filtrate derived from serum. Manyalkalizing agents, such as sodium carbonate, sodium cyanide, sodiumglycerosilicate and sodium tungstate, have been used in varyingprocesses to increase the reaction or indication speed, to increasesensitivity, to reduce turbidity or cloudiness during the reaction, orto adjust other conditions aflFecting the quality of the test.

The alkaline phosphotungstate process, because of its lack ofsensitivity and adverse side reactions that caused conditions such asturbidity, often required an excessively large serum sample. Also, thetime required for completion of the color change indicating uric acidconcentration was excessive, generally being in excess of 60 minutes.

Summary of the invention Therefore, it is an object of this invention toprovide a process that requires but a short time for completion of anindicating color change in the detection of uric acid.

A further object of this invention is to provide a process that is freeof the adverse side effects usually associated with previously knownprocesses for the detection of uric acid.

Another object of this invention is to provide a process of increasedsensitivity for detection of uric acid.

An additional object of this invention is to provide an improvedcomposition of greater sensitivity and reliability for detecting uricacid.

This invention is embodied in an improved process for detecting uricacid in a fluid containing the same, in which process a phosphotungstatereagent is reacted with the. uric acid in the fluid to yield a colorchange, the improvement comprising mixing with the fluid a colorenhancer and a sequestering agent prior to reacting the phosphotungstatereagent with the uric acid present.

The invention is also embodied in a composition for use in detectinguric acid, the composition including a color enhancer, a sequesteringagent and a phosphotungstate reagent.

In this application the term detection includes the determination of thepresence as well as of the concentration of uric acid in a fluid.

Description of the preferred embodiments Uric acid may be readily andaccurately detected in a fluid according to the novel process and withthe novel composition of this invention. The reliability of the resultsof this detection is partially dependent upon the proper preparation ofthe fluid to be tested. A commonly tested fluid for uric acid is bloodwhich is considered a complex fluid because it includes many interferingcomponents. To obtain accurate results these interfering components aretreated in such a way that they do not interfere with the uric acidindication. The process and composition of the invention will bedescribed as they are utilized for detection of uric acid in blood.However, it is understood this process and composition are not limitedto detection of uric acid in blood and may be used with other fluids.

In preparing a sample of blood for detection of uric acid, the wholeblood is allowed to separate by natural coagulation into layers of bloodcells and serum. The serum is drawn off and retained for furthertreatment. Only a small amount of serum, between about 0.3 ml. and 0.5ml., is required for detection of uric acid by the practice of thisinvention as a result of the increased sensitivity and freedom fromextraneous interference which characterizes the invention.

The serum is preferably further treated to remove pro- 3 tein. This maybe done according to the known chemical procedure in which sulphuricacid and sodium tungstate are mixed with the serum, forming aprecipitate that removes the protein. The supernatant and precipitatemay be separated by centrifuging and the supernatant drawn off forprocessing for detection of uric acid therein.

With the above noted small amount of serum utilized, between about 1 ml.and 1.5 ml. of supernatant will be produced in the protein separation.Because of the advantages of the invention, this quantity is adequate topermit detection of uric acid and determination of its concentration bythe practice of the invention therewith.

Procedures for the separation of the serum and precipitation of proteinare known and therefore, will not be further described herein.

To the supernatant formed above, a color enhancer is added to enhancethe color generated by a chromogen to be subsequently added, said colorwhen present indicating presence of uric acid. Beneficially, this colorenhancer comprises a solution of nitrogen compound such as a hydrazinesalt, for example, hydrazine sulfate, hydrazine dihydrochloride,hydrazine iodide and hydrazine tartrate, p-aminobenzoic acid,p-aminosalicyclic acid and phenylhydrazine hydorchloride.

The amount of color enhancer added is advantageously between about 1 and5 milligrams per ml. of supernatant used for testing. This amount isvariable over a wide range and is preferably limited by the colorreaction or indication appearing in a a colorless blank fluid. Acolorimeter is advantageously used to detect color changes and may beadjusted (i.e. zeroed) to compensate for a color change produced by theaddition of the color enhancer, but substantial adjustments areundesirable and may result in erroneous uric acid indications.

A sequestering agent is then added to the solution of supernatant andcolor enhancer. The sequestering agent is preferably an organic compoundselected from a group of amino carboxylic acid sequestering agents.Beneficially compounds such as ethylenedinitrillo tetraacetic acid(EDTA), tetrasodium salt; nitrilotriacetic acid (NTA):hydroxyethylenediamine triacetic acid (HEDTA), trisodium salt;diethylenetriaminepentaacetic acid (DTPA), sodium salt; di(hydroxyethyl)glycine (DEG), sodium salt; sodium pyrophosphate and combinationsthereof that do not have a free amino group are used. Although thesequestering agents listed above are in the form of a sodium salt, whichis preferred, other forms may be used. Between about 50 and 200milligrams of sequestering agent per ml. of supernatant may be used,however, this quantity is not critical and may be between about 1 and200 milligrams per ml. of supernatant used. The sequestering agent isdescribed herein as being separate from the color enhancer, however,there may be combined in a single reagent.

The solution of supernatant, color enhancer and sequestering agent ismixed thoroughly, and a chromogenic indicator such as hosphotungstatereagent is added thereto in an amount of between about 30 and 150milligrams per ml. of said supernatant. This addition is advantageouslyperformed promptly after the mixing of the three components. Thisadmixture of four components is set aside for a period of time thatpermits stabilization of any color change which may occur indicating thepresence of uric acid. For quantities of uric acid normally found inblood that is about 5 mg. percent, color stabilization is complete inabout minutes. With solutions having higher concentrations, colorstabilization is usually completed in about minutes.

A color enhancer solution for use in the above process may be preparedby dissolving one of the previously described nitrogen compound in asuitable solvent. The quantity of compound may be between about 1 and400 mg. percent and is preferably between 20 and mg. percent. Thispreferred range of concentration permits the use of a convenient ratioof about 2:1 of color enhancer solution to supernatant. Ifconcentrations in excess of about mg. percent are used, some correctionfor color in the blank is usually necessary. A sodium acetate-aqueoussolution from about 1% to saturation is a suitable solvent for the colorenhancer that also gives desirable stability to the color enhancersolution.

Sensitivity of the reaction may be increased by increasing theconcentration of color enhancer. However, an increase in color enhanceralso appears to diminish the precision obtained and as previouslymentioned, may result in color in the blank fluid.

A sequestering agent solution may be prepared by mixing one of thepreviously mentioned sequestering compounds with a suitable solvent. Theconcentration of the compound in this solution is not consideredcritical and may be between about 0.1 mg. percent and a saturatedsolution, and preferably is between about 5 and 20 grams percent.

A pH adjusting compound, preferably one that is free of interferingions, is added to the solution to be tested to adjust the pH to betweenabout 9 and 10. Advantageously, sodium tungstate is used for thispurpose, however, other compounds imparting similar pH such as sodiumhydroxide, sodium carbonate, and disodium phosphate may be used. ThesepH adjusting compounds may, of course, be in a solid state or includedin a solution that is compatible with the test system.

The color enhancer and sequestering agent may be combined to form asingle reagent. This single reagent when in an aqueous solution appearsto be stable for only about 48 hours, whereas the individual solutionsare stable for substantially longer periods. Considerable differencesmay occur between duplicates if the color enhancer solution is notaccurately measured or the combined solutions have been retained toolong. This combined reagent may also be formed as dry solid powder ortablets by evaporation or lyophilization.

Phosphotungstate chromogenic reagent solution is readily prepared bycombining sodium tungstate (molybdenum free) in an aqueous solution withorthophosphoric acid. The combined solutions are refluxed for about 2hours and cooled to room temperature. The resulting reagent should berefrigerated if retained as an aqueous solution. This reagent may beformed as solid tablets or powders by evaporation or lyophilization.

When detection of uric acid is to be carried out on an automaticanalyzing apparatus, the novel process and composition of this inventionmay be readily used. The metering of solutions and time delay may beaccording to accepted procedures with automatic equipment. For example,the serum is diluted with a saline solution and dialysed against arecipient stream containing mixed color enhancer solution andsequestering agent solution forming an admixture which is combined withphosphotungstate reagent. This combined stream is passed through a delaycoil and a determination of uric acid made with a colorimeter.

It will of course be appreciated that the order of add ing the reagentsto the fluid to be tested is not limited to that set forth in thisdescription. Also, different combinations of reagents, either in the dryform or in solutions, may be used with equally effective results.

The reagents of this invention may be placed on or in a suitable carrierthat may be dipped in the fluid to be tested, such as blood or serum,and the resulting color change read, for example, by comparison with acolor chart. Preferably, a bibulous carrier is impregnated with asolution of the reagents and then dried. The carrier is thenadvantageously coated with a protective layer that prevents a largeportion of the interfering substances from contacting the test reagent,particularly when the solution to be tested is whole blood.

When a bibulous carrier is employed the carrier material may be any of avariety of substances. For example, filter paper, wood strips, syntheticplastic fibrous materials, non-woven or woven fabrics and so on, may beutilized in this embodiment. The preferable bibulous material is filterpaper having a thickness of about 0.01 inch to about 0.02 inch.

The invention will be further understood by reference to the followingexamples which describe specific preparations of solutions and detectionof uric acid. These examples are not intended to be limitations on thescope of this invention which scope is properly defined in the appendedclaims.

EXAMPLE 1 A EDTA sequestering agent solution was prepared by dissolving50 grams of ethylenedinitrilo tetraacetic acid, tetrasodium salt indistilled water to make 1 liter. This EDTA solution was combined in a 1to 3 ratio with a previously prepared 60% sodium tungstate pH adjustingsolution. The sodium tungstate-EDTA solution formed was found to bestable at room temperature.

EXAMPLE 2 A phosphotungstate chromogenic reagent solution was preparedby dissolving 40 grams of sodium tungstate (molybdenum free) in about300 ml. of distilled water. To this solution 32 ml. of 85%orthophosphoric acid were added and the resulting solution refluxed for2 hours. The solution was cooled to room temperature and distilled waterwas added to make a volume of 1 liter. The resulting solution was storedin a refrigerator.

EXAMPLE 3 Blood samples received from 40 donors (hospital patients) wereallowed to clot and serum separated by centrifuging. A portion of serum(0.2 ml.) was withdrawn from each sample and placed in individuallyidentified test tubes containing 1.6 ml. of N sulfuric acid. Theserum-acid solutions were Well mixed and 0.2 ml. of a sodium tungstatesolution added. The solutions combined were mixed vigorously and allowedto stand for ten minutes. A precipitate formed which was separated fromthe supernatant by centrifuging. A portion of the supernatant (0.5 ml.)was added to 1 ml. of a hydrazine sulfate mg. percent) in sodium acetate(1%) solution and mixed well. Sodium tungstate-EDTA solution (4 ml.),prepared in Example 1, was added to the supernatant solution and mixedwell. Phosphotungstate reagent (1 ml.), prepared in Example 2, was addedto the supernatant solution and mixed well. The reaction solution wasallowed to stand for minutes at roon temperature. After 30 minutes thesolutions were observed to have changed from colorless solutions to bluecolored solutions, indicating the presence of uric acid. The solutionswere also observed to be free of turbidity. A blank solution includingall of the reagents but free of uric acid was compared with the testedsolutions. The blank solution was observed to be substantially colorlesswhereas the solutions containing uric acid had a blue color.Concentrations of uric acid were determined by recording the absorbanceof the solutions at 700 m with spectrophotometer. The concentration ofuric acid in the sample was determined by comparing the absorbance ofthe solutions with a graph showing absorbance relative to concentrationof uric acid that was previously prepared using solutions with knownconcentrations of uric acid.

EXAMPLE 4 The procedure of this example was the same as Example 3 withthe exception that in place of the EDTA solution a NTA solution, gramspercent, was used. The color change indication with the NTA solution wassubstantially the same as in Example 3.

EXAMPLE 5 For this example a quantity serum was prepared as described inExample 3.

The serum was divided into five equal 10 ml. portions labeled A, B, C, Dand E. Aqueous uric acid stock standard was added in varying quantities,using a microburette, representing 2 mg. percent, 4 mg. percent, 5 mg.percent, and 6 mg. percent respectively to the individual portions.

Solutions A, B, C, D and E were analyzed as described in Example 3.Recovery was calculated and it was determined 100- -2.0% of uric acidwas recovered by this method.

EXAMPLE 6 Solutions were prepared using serum and each of the compoundslisted below which are known to interfere with the detection of uricacid by conventional methods, the concentrations being graduated up tothose stated:

Mg. percent Ascorbic Acid 1.6 Sodium salicylate 100 Cysteine 1.6 Glucose500 These solutions were tested according to the procedure of Example 3,and the test results regarding identification of uric acid anddetermination of its concentration therein were in each case observed asbeing the same as the results produced when a corresponding serum samplefree of such interfering compounds was tested according to the sameprocedure.

EXAMPLE 7 The procedure of this example was the same as the procedure ofExample 3 with the exception that a HEDTA (41.3%) solution was used inplace of the EDTA solution. Substantially the same results were observedas in Example 3.

EXAMPLE 8 The procedure of this example was the same as the procedure ofExample 3 with the exception that a DTPA (40.2%) solution was used inplace of the EDTA solution. Substantially the same results were observedas in Example 3.

EXAMPLE 9 The procedure of this example was the same as the procedure ofExample 3 with the exception that a DEG (41%) solution was used in placeof the EDTA solution. Substantially the same results were observed as inExample 3.

EXAMPLE 10 The procedure of this example was the same as the procedureof Example 3 with the exception that a sodium pyrophosphate (5%)solution was used in place of the EDTA solution. Substantially the sameresults were observed as in Example 3.

EXAMPLE 1 1 A test device was prepared with two strips of cellulosepaper about .015 inch thick, inch Wide and 3 inches long. One strip wasimpregnated with a hydrazine sulfate-sodium tungstate-EDTA solution andthen dried. The second strip was impregnated with a phosphotungstatesolution and dried. The strips were individually sprayed with polyvinvlpyrrolidone and bonded together along one major face. Strips prepared inthis manner were dipped in fluids containing uric acid and a colorchange was observed that stabilized after between about 1 and 2 minutes.The strips were observed at this time and a blue color had appeared thatindicated the presence of uric acid.

The foregoing description and examples show that the present inventionprovides a novel process and composition for the detection of uric acid.Moreover, the process of this invention is more sensitive and providesreliable results in less time than currently known processes in spite ofthe presence of substances which inhibit the 7 currently knownprocesses. Furthermore, with the process and composition of theinvention a reliable and consistent test result is obtained with asmaller sample than previously required.

What is claimed is:

1. In the process for detecting uric acid in a fluid by contacting saidfluid with a phosphotungstate chromogenic reagent under alkalineconditions, the improvement comprising mixing with said fluidchromogenically enhancing amounts of a substance selected from the groupconsisting of a hydrazine salt, p-aminobenzoic acid, paminosalicylicacid and phenylhydrazine hydrochloride and an amino carboxylic acidsequestering agent prior to contacting said fluid with saidphosphotungstate chromogenic reagent.

2. A process according to claim 1 in which said fluid is blood serum.

3. A process according to claim 1 in which said color enhancer ishydrazine sulfate.

4. A process according to claim 1 in which said sequestering agent isselected from the group consisting of ethylenedinitrilo tetraaceticacid, nitrilotriacetic acid, (hydroxyethyl)ethylenediarninetriaceticacid, diethylenetriaminepentaacetic acid, di(hydroxyethyl)glycine sodiumpyrophosphate, and combinations and salts thereof.

5. A process according to claim 1 in which said sequestering agent isethylenedinitrilo tetraacetic acid.

6. A process according to claim 1 in which said sequestering agent isethylenedinitrilo tetraacetic acid, tetrasodium salt.

7. A process according to claim 1 in which said sequestering agent isnitrilotriacetic acid.

8. In a phosphotungstate chromogenic reagent a composition for detectinguric acid in a fluid the improvement which comprises the inclusiontherewith of chromogenically enhancing amounts of a substance selectedfrom the group consisting of a hydrazine salt, p-aminobenzoic acid,p-aminosalicylic acid and phenylhydrazine hydrochloride, and an aminocarboxylic acid sequestering agent.

9. A composition according to claim 8 in which said amino carboxylicacid sequestering agent is a member selected from the group consistingof ethylenedinitrilo tetraacetic acid, nitrilotriacetic acid,(hydroxyethyDethylenediaminetriacetic acid,diethylenetriaminepentaacetic acid, and di(hydroxyethyl)glycine, sodiumpyrophosphate and combinations and salts thereof.

10. A test device comprising a bibulous carrier incorporating thecomposition of claim 8.

11. A composition according to claim 8 in which said color enhancersequestering agent and phosphotungstate reagent are in aqueous solution.

References Cited Echhorn, F., et al., Journal of Clinical Pathology,vol. 14, pp. 4502 (1961).

Martel, A. E., et al., Chemistry of the Metal Chelate Compounds, pp.4995 13 (1956).

Martinek, R. G., Journal of Clinical Pathology, vol. 18, pp. 777-9(1965).

Simoes, M. S., Arquivos Portugueses De Bloquimica, vol. 6, pp. 192-7(19623).

JOSEPH SCOVRONEK, Primary Examiner E. A. KATZ, Assistant Examiner U.S.Cl. X.R. 252-408

